Multifunctional reagent for the synthesis of thiol modified oligomers

ABSTRACT

The invention relates to compounds of formula (I), wherein Z represents a hydrocarbon with 2 to 28 C atoms, wherein Z can also comprise elements N, O, P, S, Si and halogen as heteroatoms, R 1  and R 2  are identical or different sulfur protecting groups, wherein both S atoms can also form a disulfide bridge and in said case R 1  and R 2  are not present; protecting group Y 1  represents protecting group NH, protecting group NR 4 , protecting group O, CONH protecting group, protecting group OOC, protecting group S—S, —CH(protecting group O) 2  or —CR 5 (protecting group O) 2  or protecting group S; Y 2  represents —OH, —NH 2 , —NHR 3 , —NR 3 R 4 , —COOH, —COCI, —COOCO—R 6 , —CONH 2 , —CONHR 3 , —COOR 3 , —SO 3 H, —SO 3C I, —SH, —S—SR 3 , —CHO, —COR 3 , —C 2 H 3 O, halogen, —N 3 , —NH—NH 2 , —NCO, —NCS, wherein R 3  represents alkyl, heteroalkyl, aryl, cycloalkyl or a protecting group, wherein R 3  can be identical or different in groups Y 1  and Y 2 , R 4  is s protecting group, wherein R 4  can be identical or different in groups Y 1  and Y 2 , and wherein R 4  and R 3  can be identical or different, R 5  represents alkyl, aryl or cycloalkyl, wherein R 5  can be identical or different in groups Y 1  and Y 2  and R 6  represents alkyl, heteroalkyl, aryl or cycloalkyl, wherein R 6  can be identical or different in groups Y 1  and Y 2 , wherein Y 2  can also represent a group of formula (II) or formula (III), wherein X 1  represents halogen or a substituted amine, X 2  represents an alkyl, alkoxy, aryloxy radical or a cyano derivative of an alkyl, alkoxy, aryloxy radical, X 3  represents halogen, an amino function or oxygen and X 4  represents an alkyl, alkoxy, aryloxy radical or X 4  equals H if X 3 =oxygen.

TECHNICAL FIELD

This invention concerns a multifunctional reagent for the synthesis ofthiol modified oligomers.

STATE OF THE ART

Nucleic acids can be synthesized chemically or enzymatically. Dependingon the Nucleotide building block used and the reaction step for couplingit to the next nucleotide in the sequence, different procedures can bedistinguished: The phosphodiester method, the phosphotriester method andphosphoramidite method (Gait, M. J. et al., Oligonucleotide Synthesis: APractical Approach, IRL Press Oxford, 1984; Protocols forOligonucleotides and Analogs, Agrawal, S., Humana Press, New Jersey,1993). The phosphoramidite method does not use derivatives of phosphoricacid, instead it uses derivatives of phosphorous acid, so calledphosphoramidites.

The phosphoramidite method can be adapted to a solid phase method, wherethe growing nucleotide sequence is bound at a polymer carrier. Thismethod considerably simplifies the separation of the excess synthesisreagents and building blocks as well as the purification of theoligonucleotide sequence. Commercially available synthesizers operateaccording to this principle.

Nucleic acids with known nucleotide sequences have a particularapplication in the specific detection of DNA in biological samples. Insuch tests the property of single nucleotide sequences are used, namelythat they can form a double strand with their complimentary strand. Theprocess of forming a double strand is called hybridization (NucleicAcids in Chemistry and Biology, Blackburn, M. G. and Gait, J. M., OxfordUniversity Press).

The formation of a double strand can be detected, if a modified singlestranded complimentary nucleic acid is given for hybridisation to thesingle strand nucleic acid or the single stranded nucleic acid itselfhas a modification. Modifications can be i.e. fluorophores,radioisotopes or electro labels.

The application of marked targets for the detection of hybridizationevents has certain disadvantages. In the first place, the marking has tobe done before the actual measurement. This requires an additionalsynthesis step and additional working time. Furthermore it is difficultto ensure a homogenous marking of the samples. Also, stringent washingis necessary to remove non bound or unspecific bound samples afterhybridization.

Oligonucleotides and polymers in general can be immobilized on surfacesby well known methods i.e. by non covalent adsorption or by covalentcouplings onto a surface (WO 00/42217; U.S. Pat. No. 6,312,906).Especially attractive procedures to immobilize oligonucleotides onto aSiO₂ surface (glass) are based on well established silicon chemistry(Parkam et al., Biochem. Biophys. Res. Commun., 1:1-6, 1978; Lund etal., Nucl. Acids Res. 16:10861-10880, 1988). For example epoxidemodified SiO₂ surfaces can be coated by aminofunctionalizedoligonucleotides.

The chemisorption on gold was investigated more closely from 1983. Nuzzoand Allara (J. Am. Chem. Soc. 105, 4481, 1983) discovered that thiol anddisulfides adsorb on gold in ordered monolayers. The resulting covalentbond between gold and sulfur has a binding energy of 30-40 kcal/mol.Bain et al. (J. Am. Chem. Soc. 111, 321, 1989; J. Am. Chem. Soc. 111,7155, 1989) described the property of the bonding between organo sulfurcompounds and gold. The strong coordinative gold sulfur bonding advancesthe spontaneous accumulation of monolayers. Bain et al. argue that theformation of those monolayers are influenced by several factors (i.e.temperature, solvent, concentration and chainlength of the adsorbent andconcentration of salt). The adsorption is comprised of two steps: Theformation of a first monolayer coating about 80-90% of the surface isachieved within minutes, and the coating of the remaining area whichrequires a process lasting several hours. Displacements on the surface(i.e. solvent) and lateral diffusion probably play a role in thatprocess. These experiments provide the foundation for the attachment ofthiolmodified oligonucleotides.

Oligonucleotides attached onto the surface by one thiol bond, simplyexpressed by the term “Au—S-Oligonucleotide”, are unstable undermechanical stress (i. e. washing steps). The stability of theoligonucleotide on the surface is increased by multiple formedAu—S-Bondings. A very stable attachment of the oligonucleotides bringsenormous advantages for DNA Chip technology.

A variant for the attachment of DNA onto gold or platinum surfaces isprovided by the developed process of Whitesides and co-workers (Lee etal., Pure & Appl. Chem. 63, 821, 1991) to generate thiol monolayers ongold surfaces. The free thiol group of a dithiol precoated metal surface(i.e. 1.10-Decandithiol) reacts with a bromacetyl modifiedoligonucleotide.

Sulfur containing phophoramidites or polymer carriers can be used forthe production of thiol modified oligonucleotides. Examples of compoundsfor the coupling of disulfid units are the phosporamiditeDMT-O—(CH₂)₆—S—S—(CH₂)₆—O—P(OCE)(NiPr₂) or compounds of the generalformula R1-S—S—R²—O—P(OCE)(NiPr₂) (see EP 523 978). Another possibilityto couple a thiol anchor to an oligonucleotide is the use of thephosphoramidite MMT-S—(CH₂)₆—O—P(OCE)(NiPr₂), however this has thedisadvantage of the elaborate cleavage of the MMT group by AgNO₃.

In addition there are two further thiol carriers with C-3 and C-6spacers for oligonucleotide synthesis (Glen Research).

In spite of the above described state of the art there is still a needfor multifunctional thiol containing monomers able to form apolyfunctional thiol anchor, through which a stable attachment ofmolecules or polymers onto surfaces will be made possible.

DISCLOSURE OF THE INVENTION

The task of the present invention is to make thiol containing monomersavailable for the preparation of polyfunctional thiol compounds.

According to the invention the task is fulfilled by the compounds asstated in independent claim 1. Further attractive details, aspects anddevelopments of the present invention follow from the dependent claims,the description, the figures and the examples.

In this presented invention the following abbreviations and terms willbe used:

-   A: Adenine-   ACN: Acetonitrile-   Base: A, G, T, C or U-   C: Cytosine-   DMT: 4,4′-Dimethoxytrityl-   DNA: Desoxyribonucleic Acid-   E^(˜): Alternating Voltage-   EI: Electrospray Ionisation-   EtOAc: Ethylacetate-   Et₃N: Triethylamine-   f: Alternating Voltage Frequency-   Fmoc: 9-Fluorenylmethoxycarbonyl-   G: Guanine-   HPLC: Hoch Pressure Liquid Chromatography-   iPr: Isopropyl-   NMR: Nucleic Magnetic Resonance-   M: Mass-   MsCl: Mesylchloride or Methansulfonylchloride-   MeOH: Methanol-   MS: Mass Spectrometry-   mV: Millivolt-   C_(q): Quarternary Carbon-   C_(arom): Aromatic Carbon-   H_(arom): Aromatic Hydrogen-   OD₂₆₀: Optical Density (260 nm)-   OCE: Cyanoethoxy-   Oligomer: Equivalent to Nucleic Acid Oligomer-   Oligonucleotide: DNA-, PNA- or RNA-Fragment with no specified    chainlength of bases-   PNA: Peptide Nucleic Acid (—NH—(CH₂)₂—N(COCH₂-Base)-CH₂CO; synthetic    DNA or RNA in which the sugar phosphate unit is substituted by an    amino acid. PNA can be hybridized with DNA or RNA).-   RNA: Ribonucleic Acid-   R_(f): Retention at TLC relative to the solvent front-   rms: root mean square-   RP: Reverse Phase-   s: Singlet-   SPR: Surface Resonance Spectroscopy-   T: Thymine-   TCL: Thin Liquid Chromatography-   U: Uracile-   v: velocity of feed

Every formula is to be interpreted in a way such that the correspondingchiral enantiomers are included.

The presented invention includes compounds of the formula (I)

Where Z is a hydrocarbon of 2 to 28 C atoms, where Z can also includeheteroatoms of the elements N, O, P, S, Si and halogen, R¹ and R² arethe same or different H or sulfur protecting groups, where both S atomscould also form a disulfide bridge and in which case R¹, R² would notexist, protecting-group-Y¹, is protecting-group-NH,protecting-group-NR⁴, protecting-group-O, CONH-protecting-group,protecting-group-OOC, protecting-group-S—S, —CH(protecting-group-O)₂, or—CR⁵(protecting-groupO)₂ or protecting-group-S, Y² is —OH, —NH₂, —NHR³,—NR³R⁴, —COOH, —COCl, —COOCO—R⁶, —CONH₂, —CONHR³, —COOR³, —SO₃H, —SO₃Cl,—SH, —S—SR³, —CHO, —COR³, —C₂H₃O, halogen, —N₃, —NH—NH₂, —NCO, —NCS,where R³ is alkyl, heteroalkyl, aryl, cycloalkyl or a protecting group,where R³ can be the same or different in the groups Y¹ and Y², R⁴ is aprotecting group, where R⁴ can be the same or different in the groups Y¹and Y², and where R⁴ and R³ can be the same or different, R⁵ is alkyl,aryl, cycloalkyl, where R⁵ can be the same or different in the groups Y¹and Y² and R⁵ is alkyl, heteroalkyl, aryl, or cycloalkyl, where R⁶ canbe the same or different in the groups Y¹ and Y², where Y² can also be agroup of the formula (II) or (III),

Where X¹ is a halogen or a substituted amine, X² is alkyl, alkoxy,aryloxy or a cyano derivative of alkyl, alkoxy, aryloxy, X³ is ahalogen, an amino group or oxygen and X⁴ is alkyl, alkoxy, aryloxy or X⁴is H where X³ is oxygen.

The invented compounds are substances consisting of at least fourfunctional groups, of which two are thiols (R¹ , R²═H), thioethers ordisulfides, where both sulfur atoms can be joined together to form adisulfide bridge and R¹, R² do not exist. Y¹ is a functional group witha protecting group. Y² is a functional group, which serves amongst otherthings for the activation of the invented compounds for chemicalreactions. Such chemical reactions are for example a polymerisation oran attachment to a polymeric carrier material. Where Y² can be analready activated functional group or a to be activated functionalgroup. The fundamental body Z is a structure of hydrocarbon consistingof 2 to 28 atoms, where Z can also include heteroatoms of the elementsN, O, P, S, Si and halogen. The compounds of formula (I) have at leastone protecting group.

The invented compounds can be used for the directed and definedconstruction of oligomers, or polymers with an exactly defined number ofsulfur atoms. For which the selective cleavage of a protecting group isa necessary prerequisite. The chemical reaction of the monomers must notinfluence the integrity of the protecting group. The existing R¹, R² atthe sulfur must be chemically stable during polymerisation whichincludes the activation of the functional group and the cleavage of theprotecting group: This means the protecting group at Y¹, has to beorthogonally or selectively cleavable to R¹, R² at the sulfur.

Should R¹ and R² not exist and as a result the two sulfur atoms arejoined together to form a disulfide bridge, the fact that the disulfideunit is not located in the backbone of the polymer represents a specialadvantage in that a cleavage of the disulfide bridge/s does not cause adestruction of the polymer.

Protecting groups can be amongst others triphenylmethyl-,t-butoxycarbonyl-, benzyl-, 2,4dinitrophenyl-,9-fluorenylmethoxycarbonyl-, allyloxycarbonyl-, benzyloxymethyl-,acetyl-, 4azidobenzyloxycarbonyl-, acetamidomethyl-, 1-adamantyl-,1-adamantyloxycarbonyl-, anisyl, benzamidomethyl-,biphenyldimethylsilyl-, 2,4dimethylthiophenoxycarbonyl-,1-methyl-1-(4-biphenyl)ethoxycarbonyl-, benzothiazole-2-sulfonyl-,t-butoxymethyl-, benzoyl-, benzyloxycarbonyl-, cyclohexan-1,2-diacetal-,cyclohexyl-, 2-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-,1-methyl-1-(3,5-dimethoxyphenyl)ethoxycarbonyl-, diethylisopropylsilyl-,1,3-dithianyl-2-methyl-, 2,4dimethoxybenzyl-, dithianylmethoxycarbonyl-,dimethoxytrityl-, p,p′-dinitrobenzhydryl-, 2,4-dinitrophenyl-,2,4dimethylpent-3-yloxycarbonyl-, 2-(diphenylphosphino)ethyl-,9-fluorenylmethyl-, levulinoyl-, p-methoxybenzensulfonyl-,2,6-dimethoxy-4-methoxybenzensulfonyl-, monomethoxytrityl-,methoxyphenylsulfonyl-, mesitylensulfonyl-, o-nitrobenzyl-,2-[2-(benzyloxy)ethyl]benzoyl-, 3-(3-pyridyl)allyloxycarbonyl-,2,2,5,7,8-pentamethylchroman-6-sulfonyl-, pivaloyloxymethyl-,t-butyidimethylsilyl-, t-butyldiphenylsilyl-,2,2,2-trichloro-1,1-dimethylethyl-, trifluoroacetyl-, triisobutylsilyl-,2,4,6-trimethylbenzyl-, trimethoxybenzyl-, p-toluensulfonyl- orbenzyloxycarbonyl-. Further protecting groups can be found in Greene, T.W., Protective Groups in Organic Synthesis, Wiley-Interscience, 1999.

R¹ and R² are identical or disparate sulfur protecting groups, which canbe amongst others derivatives of benzyl, triphenylmethyl, substitutedmethyl, benzoyl-, trifluoracetyl- or t-butoxycarbonyl-groups. Inaddition the thiols can be protected as disulfides i.e. S-ethylsulfides,S-phenyldisulfides or as thiocarbamates. Further protecting groups canbe found in Greene, T. W., Protective Groups in Organic Synthesis,Wiley-Interscience, 1999.

All of the invented compounds can be incorporated in oligomers asmonomer units.

The invented compounds can be singly or multiply attached, independentlyof the position in the nucleotide oligomers, peptide oligomers or othermolecules. Therefore for example the attachment of any molecule and alsopolymers onto a surface or at a polymer carrier using a polythiol anchoris possible. By the activation of the polyfunctional thiol anchormultiple attachments of the molecule onto the surface (i.e. goldsurface) or to another polymer carder are formed. For this specialpreactivated surfaces (i.e. surfaces activated by aldehyde or maleimide)or special polymer compounds having thiol reactive groups for examplepeptides, proteins, PNA, RNA, LNA (locked nucleic acids) can be used.Those invented compounds that are attached at molecules or in particularat polymers can also be used for the attachment of markers (labels) orother functionalities at molecules or in particular at polymers.

An advantage of the invented compounds is the possibility to incorporatemultiple SH groups into oligomers thereby generating higher stabilitythrough the immobilization of the oligomers onto a surface. Furtheradvantage is the possibility to couple various molecules i. e. polymers,peptides, proteins or oligonucleotides at a polymer with multipleSH-groups.

Y² can also be coupled direct or via a linker to a solid carriermaterial i.e. CPG (controlled pore glass), microbeads, polymers (i.e.polystyrene) or membranes. To synthesize nucleic acids, the inventedcompounds will normally be attached to an solid carrier via anaminoalkyl (LCM=long chain alkyl amine).

The preferred in the context of the presented invention reactivephosphor intermediates can be singly or multiply incorporated at the 3′end, in the middle and at the 5′ end of an oligonucleotide. Byactivation a multiple polyfunctional thiol anchor can be liberated,which can be used for a multiple attachment for example of anoligonucleotide onto a surface; onto preactivated surfaces (i.e.aldehyde, maleimide), or at other polymer compounds (i.e. proteins, PNA,RNA, LNA) having groups reactive to thiols. Furthermore thispolyfunctional polythiol anchor can be used for the directed multipleattachment of markers and ligands to molecules or polymers. Markers andligands may be for example ezymatic, chromogene, fluorogene,radioactive, chemiluminiscent labels, in nucleic acids oligomersintercalating agents, metals, metal ions, drugs, hormones, proteins,peptides, nucleolytic or proteolytic agents, especially binding agents(like i.e. biotin, antigenes, haptenes, antibodies, receptors) and othercompounds of biological interest, which i.e. influence the transportthrough biological membranes or change the solubility ofoligonucleotides. There are known procedures, which make it possible tocouple these ligands and markers to thiol groups such as for example bymaleimides. aldehydes and halogenacetyl compounds (Means, G. M. and R.E. Feeney, Chemical Modification of Proteins, Holden-Day Inc., 1971;Feeney, R. E., Int. J. Peptide Protein Res., 29: 145-161, 1987; Eritja,R. et al., Tetrahedron, 47, 4113-4120, 1991).

The bonding between the surface and the oligonucleotide via one thiolanchor, simplified as surface-S-oligonucleotide, is not stable tomechanical stress, for example during washing steps. One of theadvantages of the presented invention is the possibility to couple apoly anchor at an oligonucleotide, such that the attachment of theoligonucleotide to gold is optimised by several Au—S-bondings. In whichcase the conditions for the attachment onto the surface for example theconcentration of salt used, the applied potential at the surface or thekind of premodificated surface are crucial. Furthermore thiols alreadydeposited on the surface can be displaced by this poly anchor.

Furthermore the deprotection of the sulfur protecting group by AgNO3 isavoided by the use of DMT as the protecting group. The DMT protectinggroup can be cleaved by mild acid treatment, which is compatible witholigonucleotide chemistry. The presented invented compounds can be usedunder the usual standard conditions for oligonucleotide chemistry.

The phosphorous containing compounds include intermediates that can beused in the H-phosphonate, phosphotriester, phosphorchloridite andphosphoramidite methods of oligonucleotide synthesis. Furthermore theseintermediates can include phosphodiester analogs such as methylphosphonates, methyl phosphates, phosphorthioates and phosphoramidites(EP 0 523 978), for modifications at 5′-, 3′ end and/or in the sequence.

According to a preferred embodiment of the invention Y² is equivalent toformula II or III, in which X¹ is a halogen and X²is methyl or R⁷O—,where R⁷ is alkyl, cycloalkyl, aryl or a cyano derivative of alkyl,aryl, or X² is equivalent to R⁷O— and X¹ is equivalent to —NR⁸R⁹, whereR⁸ and R⁹ are independently from each other alkyl, heteroalkyl,cycloalkyl, aryl or R⁸ and R⁹ are joined together to form with the Natom a cyclic structure of 4 to 7 C atoms, in which one C atom of thecyclic structure can be replaced by O or S, or X³═O⁻ and X⁴═H or isR¹⁰O—, in which R¹⁰ is a protecting group.

The presented invented compounds can also be bound to a carrier material(solid support), if Z possesses a free or a protected OH function. Awide selection of carrier materials can be used, for instance silica,Porasil C, polystyrene, Controlled Pore Glass (CPG), Kieselgur,poly(dimethylacrylamide), poly(acrylmorpholino), Cellulose, Fractosil500. Depending on the type of carrier materials differentfunctionalities for the anchor are used. Substituted alkyl or aryl silylcompounds are used for silicon carrier materials like Silica and glassto form a siloxan or siloximine anchor. Ethers, esters, amines, amides,sulfides, sulfones and phosphates can be used by organic polymers.

In the case that Y² is a group of the formula (III), in which X³ isequivalent to O⁻ and X⁴ is equivalent to H, the above mentionedcompounds represent H-phosphonates and are employed in the H-phosphonatemethod for the oligonucleotide synthesis (Sinha und Cook, Nucleic AcidsResearch (1988) 16:2659-2669). H-phosphonates may be converted tophosphit diesters, phosphorothioates, or phosphoramidates, as soon theyare incorporated at the 5′ end of the oligonucleotide (Miller et al.,Nucleic Acids Res. (1983) 11:5189-5204, Eckstein, Ann. Rev. Biochem.(1985) 54:367-402).

Accordingly, the above mentioned compounds in which Y² is a group of theformula (III) in which X³ is equivalent to O⁻ and X⁴ is equivalent toR¹⁰O— can be used in the phosphotriester method for oligonucleotidesynthesis (Garegg, et al., Chemica Scripta (1985) 26:5).

The compounds in which Y² represents a group of the formula (II), inwhich X¹ is equivalent to chlorine and X²is equivalent to R⁷O—, arephosphochloridites and are used in the phosphochloridite technique foroligonucleotide synthesis (Wada et al., J. Org. Chem. (1991)56:1243-1250).

The phosphoramidites in which Y² is equivalent to the above formula (II)are especially preferred for the purpose of the presented invention.

R¹ and R² are the same or different H or sulfur protecting groups.Preferred sulfur protecting groups are for example trityl,4,4′-dimethoxytrityl, 4-monomethoxytrityl, 9-fluorenylmethyl (Ponsati,B., et al., Tetrahedron, 46, 8255-8266, 1990),9-fluorenylmethoxycarbonyl, 2,4-dinitrophenylethyl,2,4,6-trimethoxybenzyl (Munson, M. C. et al., J. Org. Chem., 57,3013-3018, 1992), 4-methoxybenzyl and allyloxycarbonylaminomethyl(Kimbonguila, A. M., et al., Tetrahedron 55, 6931-6944, 1999).Especially preferred is 4,4′-dimethoxytrityl. Further protecting groupscan be found in Lloyd-Williams, P. et al., Chemical Approaches to theSynthesis of Peptides and Proteins, New York, CRC Press. The sulfurprotecting groups trityl and acetamidomethyl can be cleaved by iodineoxidation (Kamber et al., Helvetica Chimica Acta, Vol. 63, No. 96,899-915, 1980). The sulfur protecting groups 4,4′-dimethoxytrityl and4-monomethoxytrityl can be cleaved by AgNO₃ in methanol (Huang, Z. andBenner, S. A., Synlett, 83-84, 1993). The 4,4′-dimethoxytrityl sulfurprotecting group can also be cleaved under mild acid conditions (i.e. 2%dichloro acetic acid in dichloromethane), that is compatible to theoligonucleotide synthesis. The great variety of sulfur protecting groupsoffers the opportunity to select orthogonal protecting groups , whosedeprotection conditions are compatible to the oligonucleotide synthesisto introduce different labels.

According to a preferred embodiment of the presented invention the restR⁷ represents a base labile protecting group.

In a particularly preferred embodiment R⁷ is a base labile protectinggroup selected from β-cyanoethyl, β-nitroethyl, 2,2,2-trichlorethyl,methyl, 1,1-dimethyl-2,2,2-thrichlorethyl, 2,2,2-tribromethyl, benzyl,o-chlorphenyl, p-nitrophenylethyl, 2-methylsulfonylethyl and1,1-dimethyl-2-cyanoethyl.

According to an especially preferred embodiment of the presentedinvention where R⁷ is a base labile protecting group R⁸ and R⁹ areindividually alkyl consisting of 1 to 16 C atoms, cycloalkyl consistingof 3 to 8 C atoms, aryl consisting of 6 to 20 C atoms; or R⁸ and R⁹ arejoined together to form with a N atom a cyclic structure with 4 to 7 Catoms, in which a C atom of the cyclic structure can be replaced by O orS. Moreover it is especially preferred, if R⁸ and R⁹ are independentlyfrom each other alkyl consisting of 1 to 6 C atoms. Also especiallypreferred is where R⁸ and R⁹ are isopropyl, butyl, hexyl, nonyl,dodecyl, hexadecyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclooctyl,phenyl, tolyl, benzyl, xylyl, naphthyl, morpholino, piperidinyl orthiomorpholino.

Further protecting groups R⁸ and R⁹ are listed in Green, T. W.,Protective Groups in Organic Chemistry, New York: Wiley & Sons, 1981.

According to a preferred embodiment of the presented invention, Z is ahydrocarbon structure consisting of 2 to 28 C atoms, in which Z alsoincludes the heteroatoms of the elements N, O, P and S.

According to a further preferred embodiment of the presented invention,Z is a hydrocarbon structure consisting of 2 to 28 C atoms, in which Zalso includes the heteroatoms of the elements N, O and P.

According to a further preferred embodiment of the presented invention,Z is a hydrocarbon structure consisting of 2 to 28 C atoms, in which Zalso includes the heteroatoms of the elements N and O.

According to a further preferred embodiment of the presented invention,Z is a hydrocarbon structure consisting of 2 to 28 C atoms, where Z canalso include the heteroatoms of the elements N and O, in which theelements N and O are solely present as part of an amide bond.

According to a further preferred embodiment of the presented invention,Z is a hydrocarbon structure consisting of 2 to 28 C atoms, in which Zalso includes the heteroatoms of the elements P and O.

According to a further preferred embodiment of the presented invention,Z is a hydrocarbon structure consisting of 2 to 28 C atoms, where Z canalso include the heteroatoms of the elements P and O, in which theelements P and O are solely present as part of a phospor diester bond.

According to a further preferred embodiment of the presented invention,Z is a hydrocarbon structure consisting of 2 to 8 C atoms.

According to an especially preferred embodiment of the presentedinvention, Z is a hydrocarbon structure consisting of 4 C atoms and 6 Hatoms.

According to an especially preferred embodiment of the presentedinvention compounds of the formula

are provided, where A¹, A², A³, A⁴, A⁵, A⁶ are the same or differentalkyl of 1-22 C atoms, heteroalkyl of 1-22 C atoms, cycloalkyl of 1-22 Catoms, H, protecting-group-Y¹ or group Y², where A¹, A², A³, A⁴, A⁵, A⁶can also include protecting-group-Y¹ and group Y² and whereprotecting-group Y¹ as well as group Y² is present at least once.

The compounds according to formula (IV) possess at least two furtherfunctional groups besides both S atoms. Moreover the substitutes A¹, A²,A³, A⁴, A⁵ and A⁶ can be a functional group. A¹, A², A³, A⁴, A⁵ and A⁶can also be alkyl of 1-22 C atoms, heteroalkyl of 1-22 C atoms or H, inthe case that out of the six substitutes A¹, A², A³, A⁴, A⁵ and A⁶ notless than two of the substitutes are functional groups, at least themissing functional group(s) must be bound to the above definedheteroalkyl(s). At least one of these functional groups is protected bya protecting group.

Preferred are compounds, where A² and A⁴ are equal toprotecting-group-Y¹ or Y², where A², A⁴ can also includeprotecting-group-Y¹ and group Y² and where protecting-group-Y¹ as wellas group Y² are present at least once. In the case that compounds of thestructure (IV) are present, where A¹, A³, A⁵ and A⁶ are alkyl of 1-22 Catoms, heteroalkyl of 1-22 C atoms, cycloalkyl of 1-22 C atoms or H. A²and A⁴ are functional groups or include a functional group. Thesubstitute, which is not a functional group, is a heteroalkyl of 1-22 Catoms or a cycloheteroalkyl of 1-22 C atoms including at least onefunctional group. At least one of these functional groups is protectedby a protecting group. Should A² or A⁴ include two functional groups,the substitute not including a functional group can be an alkyl of 1-22C atoms, heteroalkyl of 1-22 C atoms, cycloalkyl of 1-22 C atoms or H.

Especially preferred are compounds, where A², A⁴ are identical toprotecting-group-Y¹, Y², where A² is not identical to A⁴. In that casecompounds of the structure (IV) are present, where A¹, A³, A⁵ and A⁶ arealkyl of 1-22 C atoms, heteroalkyl of 1-22 C atoms, cycloalkyl of 1-22 Catoms or H. A² and A⁴ are functional groups. At least one of thesefunctional groups is protected by a protecting group.

Especially preferred are compounds, where A¹, A³, A⁵ and A⁶ areidentical to H. In that case compounds of the structure (IV) arepresent, where A¹, A³, A⁵ and A⁶ are H and A² and A⁴ are a functionalgroup. At least one of these functional groups is protected by aprotecting group.

Also preferred are compounds of the structure (IV), whereprotecting-group-Y¹ is identical to protecting-group-OOC,protecting-group-O, protecting-group-S, protecting-group-NH orprotecting-group-NR^(y) and Y² is identical to COOH, COOR^(x), OH,OR^(x), SH, SR^(x), NH₂, NHR^(x), NR^(x)R^(y), where R^(x) is aprotecting group and R^(y) is an alkyl of 1-15 C atoms, an aryl of 1-14C atoms, a cycloalkyl of 1-15 C atoms, a heteroalkyl of 1-15 C atoms, aprotecting group or a group of the formula (II) or (III).

Most especially preferred are compounds of the structure (IV), where A²is identical to protecting-group-O, protecting-group-OOC orprotecting-group-NH, where A⁴ is identical to COOH, a group of theformula (II) or (III) or protecting-group-NH, and R¹═R²=DMT or both Satoms are joined together to form a disulfide bridge and in that caseR¹, R² do not exist.

According to an especially preferred embodiment of the presentedinvention compounds of the formula

are provided, where D¹, D², D³, D⁴, D⁵, D⁶ are the same or differentalkyl of 1-22 C atoms, heteroalkyl of 1-22 C atoms, cycloalkyl of 1-22 Catoms, H, protecting-group-Y¹ or group Y², where D¹, D², D³, D⁴, D⁵, D⁶can also include protecting-group-Y¹ and group Y² and whereprotecting-group-Y¹ as well as group Y² is present at least once.

In addition to containing both S groups, compounds according to formula(V) have at least two further functional groups. Moreover thesubstitutes D¹, D², D³, D⁴, D⁵ and D⁶ can be a functional group. D¹, D²,D³, D⁴, D⁵ and D⁶ can also be alkyl of 1-22C atoms, heteroalkyl of 1-22C atoms, cycloalkyl of 1-22 C atoms or H, in the case that out of thesix substitutes D¹, D², D³, D⁴, D⁵ and D⁶ not less than two of thesubstitutes are functional groups, at least the missing functionalgroup(s) must be bound to the above defined heteroalkyl(s). At least oneof these functional groups is protected by a protecting group. R¹ and R²are as defined above.

Also preferred are compounds of the structure (V), where D¹, D³ and D⁵are alkyl of 1-22 C atoms, heteroalkyl of 1-22 C atoms, cycloalkyl of1-22 C atoms or H. The substitutes D², D⁴, D⁶ can be a functional group.D², D⁴ and D⁶ can also be alkyl of 1-22 C atoms, heteroalkyl of 1-22 Catoms or cycloalkyl of 1-22 C atoms or H, in the case that out of thethree substitutes D², D⁴ and D⁶ not less than two of the substitutes arefunctional groups, at least the missing functional group(s) must bebound to one of the above defined heteroalkyl(s). At least one of thesefunctional groups is protected by a protecting group.

Also preferred are compounds of the structure (V), where D² or D⁴ or D⁶is identical to protecting-group-Y¹ or Y² and D² or D⁴ or D⁶ include thegroups protecting-group-Y¹ or Y², where protecting-group-Y¹ as well asgroup Y² are present at least once.

Also preferred are compounds of the structure (V), where D¹, D², D³ andD⁵ are identical to H and D⁴ and D⁶ include the groupsprotecting-group-Y¹ and Y².

Also preferred are compounds of the structure (V), whereprotecting-group-Y¹ is identical to protecting-group-OOC,protecting-group-O, protecting-group-S, protecting-group-NH orprotecting-group-NR^(y) and Y² is identical to COOH, COOR^(x), OH,OR^(x), SH, SR^(x), NH₂, NHR^(x) or NR^(x)R^(y), where R^(x) is aprotecting group and R^(y) is an alkyl of 1-15 C atoms, an aryl of 1-14C atoms, a cycloalkyl of 1-15 C atoms, a heteroalkyl of 1-15 C atoms, aprotecting group or a group of the formula (II) or (III).

Also preferred are compounds of the structure (V), where D¹, D², D³ andD⁵ are identical to H and D⁴ and D⁶ are protecting-group-Y¹, Y².

Also preferred are compounds of the structure (V), where D¹, D², D³, D⁴and D⁵ are identical to H and D⁵ is heteroalkyl of 1-22 C atoms orcycloheteroalkyl of 1-22 C atoms, where D⁶ include the groupsprotecting-group-Y¹ and Y², where R¹═R²=DMT or both S atoms are joinedtogether to form a disulfide bridge and in that case R¹, R² do notexist.

Also especially preferred are compounds of the structure (V), where D¹,D², D³ and D⁵ are H. D⁴ or D⁶ are a functional group. The substitutethat is not a functional group is a heteroalkyl of 1-22 C atoms or acycloheteroalkyl of 1-22 C atoms with at least one functional group. Atleast one of these functional groups is protected by a protecting group.

Also especially preferred are compounds of the structure (V), where D¹,D², D³ and D⁵ are H. The substitutes D⁴ and D⁶ are alkyl of 1-22 Catoms, heteroalkyl of 1-22 C atoms, cycloalkyl of 1-22 C atoms or H,with the restriction that at least two functional groups are boundamongst D⁴ and D⁶. At least one of these functional groups is protectedby a protecting group.

Especially preferred are the functional groups COOH, COOR^(x), OH,OR^(x), SH, SR^(x), NH₂, NHR^(x) and NR^(x)R^(y), where R^(x) representsa protecting group and R^(y) is alkyl of 1-15 C atoms, heteroalkyl of1-15 C atoms, aryl of 1-14 C atoms, cycloakyl of 1-15 C atoms, or aprotecting group, which can be cleaved independently of R^(x3) or agroup of the formula (II) or (III).

Most especially preferred are compounds of the structure (V), where D¹,D², D³, D⁴ and D⁵ are H. D⁶ is a hteroalkyl of 1-22 C atoms or acycloheteralkyl of 1-22 C atoms including a total of at least twofunctional groups, at least one of which is protected by a protectinggroup. The functional groups are a group of formula (II) or OR^(x),COOR^(x), COOH or NHR^(x), where R^(x) is DMT, Fmoc or an alkyl of 1-22C atoms. R¹═R²=DMT or R¹ and R² do not exist, if both S atoms are joinedtogether to form a disulfide bridge.

According to an especially preferred embodiment of the presentedinvention compounds of the formula

are provided, where B¹, B², B³ and B⁴ are identical or different alkylof 1-22 C atoms, heteroalkyl of 1-22 C atoms, cycloalkyl of 1-22 Catoms, H, protecting-group-Y¹ or group Y², where B¹, B², B³and B⁴ canalso include protecting-group-Y¹ and group Y² and whereprotecting-group-Y¹ as well as group Y² is present at least once.

In addition to containing both S groups, compounds of formula (VI) haveat least two further functional groups. Further the substitutes B¹, B²,B³, B⁴ can be a functional group. B¹, B², B³ and B⁴ can also be alkyl of1-22 C atoms, heteroalkyl of 1-22 C atoms, cycloalkyl of 1-22 C atoms orH, in the case that out of the four substitutes B¹, B², B³, B⁴ not lessthan two of the substitutes are functional groups, at least the missingfunctional group(s) must be bound to the above defined heteroalkyl(s).At least one of these functional groups is protected by a protectinggroup.

Especially preferred are compounds of the structure (VI), where B² or B⁴is identical to protecting-group-Y¹ or Y² and either B² or B⁴ includethe groups protecting-group-Y¹ or Y², where protecting-group-Y¹ as wellas group Y² are present at least once.

Also especially preferred are compounds of the structure (VI), where B¹,B² and B³ are identical to H and B⁴ include the groupsprotecting-group-Y¹ and Y².

Also especially preferred are compounds of the structure (VI), where B⁴is a heteroalkyl of 1-22 C atoms or a cycloheteroalkyl of 1-22 C atoms,where B⁴ includes the groups protecting- group-Y¹ and Y², whereprotecting-group-Y¹ is identical to protecting-group-OOC,protecting-group-O or protecting-group-NH and Y² is identical to COOH,COOR^(x), OR^(x), NHR^(x) or a group of formula (II), where R^(x) isidentical to DMT or Fmoc and R¹═R²=DMT or both S atoms are joinedtogether to form a disulfide bridge and in that case R¹, R² do notexist.

Also especially preferred are compounds of the structure (VI), where B¹and B³ are alkyl of 1-22 C atoms, heteroalkyl of 1-22 C atoms,cycloalkyl of 1-22 C atoms or H. B² and B⁴ are a functional group. Atleast one of these functional groups is protected by a protecting group.

Also especially preferred are compounds of the structure (VI), where B¹and B³ are alkyl of 1-22 C atoms, heteroalkyl of 1-22 C atoms,cycloalkyl of 1-22 C atoms or H. B² or B⁴ are functional groups. Thesubstitute that is not a functional group is a heteroalkyl of 1-22 Catoms or a cycloheteroalkyl of 1-22 C atoms with at least one functionalgroup. At least one of these functional groups is protected by aprotecting group.

Also especially preferred are compounds of the structure (VI), where B¹and B³ are alkyl of 1-22 C atoms, heteroalkyl of 1-22 C atoms,cycloalkyl of 1-22 C atoms or H. The substitutes B² and B⁴ are alkyl of1-22 C atoms, heteroalkyl of 1-22 C atoms, cycloalkyl of 1-22 C atoms orH, with the restriction that at least two functional groups are boundamongst B² and B⁴. At least one of these functional groups is protectedby a protecting group.

Also especially preferred are compounds of the structure (VI), where B¹,B², B³ are H. B⁴ is a heteroalkyl of 1-22 C atoms or a cycloheteroalkylof 1-22 C atoms including a total of at least two functional groups, ofwhich at least one is protected by a protecting group. The functionalgroups are COOH, COOR^(x), OH, OR^(x), SH, SR^(x), NH₂, NHR^(x),NR^(x)R^(y), where R^(x) represents a protecting group and R^(y) isalkyl of 1-15 C atoms, heteroalkyl of 1-15 C atoms, aryl of 1-14 Catoms, cycloalkyl of 1-15 C atoms or a protecting group, which can becleaved independently of R^(x) or a group of formula (II).

Also especially preferred are compounds of the structure (VI), where B¹,B², B³ are H. B⁴ is a heteroalkyl of 1-22 C atoms or a cycloheteroalkylrest of 1-22 C atoms including a total of at least two functionalgroups, of which at least one is protected by a protecting group. Thefunctional groups are a group of formula (II) or OR^(x), COOR^(x), COOHor NHR^(x), where R^(x) is DMT, Fmoc or an alkyl of 1-22 C atoms.R¹═R²=DMT or R¹ and R² are not present, if both S atoms are joinedtogether to form a disulfide bridge.

The presented invention includes also the application of the inventedcompounds for the modification of oligomers. In addition the presentedinvention also includes the application of the invented compounds forthe immobilisation of modified oligomers on surfaces. Furthermore thepresented invention also includes the application of the inventedcompounds for the conjugation of enzymatic, chromogene, fluorogene,radioactive or chemiluminiscent labels, substances intercalating innucleic acids, metals, metal ions, hormones, proteins, peptides,nucleolytic and proteolytic agents, biotin, antigens, haptens,antibodies or receptors to molecules or oligomers. Finally the inventionalso includes the application of the invented compounds for theautomatic synthesis of oligomers.

Within the described applications of the invented compounds it isespecially preferred that the oligomers used are oligonucleotides,polypeptides, PNA or LNA (Locked Nucleic Acid).

The synthesis of oligonucleotides which are modified with the inventedcompounds takes place in solution or preferably at solid phase, ifnecessary using an automated synthesiser.

A SHORT DESCRIPTION OF THE DRAWINGS

FIG. 1 synthesis scheme according to example 4;

FIG. 2 SPR kinetic for the immobilization of oligonucleotides onto agold surface according to example 13;

FIG. 3 tests for stability of an oligonucleotide on a gold surfaceaccording to example 13;

FIG. 4, FIG. 4.1 and FIG. 4.2 show the results of a square wavevoltammetric measurement to prove the hybridizability of theoligonucleotides on a gold surface according to example 13;

-   -   FIG. 4.3 and FIG. 4.4 show the results of cyclovoltammetric        measurements to quantify the hybridizability of the        oligonucleotides on a gold surface according to example 13;

FIG. 5 Shows the extension of the surface coverage by oligonucleotideson a gold surface by integration of the peak areas of thecyclovoltammograms according to example 13;

FIG. 6 synthesis scheme according to example 15;

FIG. 7 synthesis scheme according to example 16;

FIG. 8 synthesis scheme according to example 17;

FIG. 9 synthesis scheme according to example 18.

POSSIBILITIES FOR REALISING THE INVENTION EXAMPLE 1 Synthesis of3-O-(4,4′-dimethoxytrityl)-1,4-bis-(4,4′-dimethoxitrityl)-sulfanyl-butan-2-ol

In an argon atmosphere 2.0 g (12.9 mmol) of 1,4-Dithio-butan-2,3-diol isdissolved by stirring in 35 ml anhydrous pyridine in a round-bottomed250 ml flask. 15.3 g (45.15 mmol) of DMT-Cl (4,4′-DimethoxytritylChloride) is added to the clear solution. After 2 hours stirring at roomtemperature the reaction mixture is heated to 50° C. and is stirredovernight. Thereafter MeOH (2 ml) is added and stirred for 10 min. Afterconcentration in high vacuum the residue is dissolved in 200 ml DCM andis then extracted once with a 1 mol/l NaHCO₃ solution and once with aNaCl solution. The organic phase is dried with Na₂SO₄, filtered andconcentrated. For purification of the raw material silica gel 60 is usedfor chromatography (eluent: ethylacetate/n-heptane/1% Et₃N). The productcontaining fractions are collected and the solvent is evaporated todryness under vacuum. TLC (silica gel, EtOAc/n-heptane=1:2, +1% Et₃N):R_(f)=0.20.

5.90 g (43.2% of the theoretical yield) of a yellowish foaming residueis obtained. ¹³C-NMR (CD₃Cl) δ (ppm): 33.21 (C-4), 35.82 (C-1), 55.17(OCH₃), 65.50 and 65.82 (C-2 and C-3), 71.52 and 75.02 (S—C_(q) DMT),87.03 (O—C_(q) DMT), 113.01, 126.32, 127.71, 129.30, 131.03, 136.21,137.13, 145.25, 145.76, 146.10, 157.72, 158.61 (C_(arom) DMT). ¹H-NMR(CD₃Cl) δ (ppm): 2.05-2.21 (m, 4H, CH₂-1 und CH₂-4), 3.17 (m, 1H, H-3),3.65 (m, 1H, H-2), 6.72-7.38 (m, 39H_(arom) DMT).

EXAMPLE 2 Synthesis of3-O-(4,4′-Dimethoxytrityl)-1,4-bis-(4,4′-dimethoxitrityl)-sulfanyl-butan-2-O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite

In an argon atmosphere 503 mg (0.47 mmol) of3-O-(4,4′-dimethoxytrityl)-1,4-bis-(4,4′-dimethoxitrityl)sulfanyl-butan-2-olare dissolved in 4 ml anhydrous ACN. The solution is cooled with ice and753.2 μl (4.4 mmol) of N,N′-diisopropylethylamine are added dropwisewhile stirring. 295 μl (1.32 mmol) ofchlor-(2-cyanoethoxy)(diisopropylamino)-phosphine are added dropwiseusing a syringe. After 1.5 hours stirring at room temperature thereaction mixture is diluted with 30 ml DCM and is extracted once with a1 mol/l NaHCO₃ solution and once with a saturated sodium chloridesolution. The organic phase is dried with Na₂SO₄, filtered andconcentrated. For purification of the raw material silica gel is usedfor chromatography (eluent: gradient ethlyacetate/n-heptane 3:1 to 2:1in the presence of 1% Et₃N). Both diastereomeres can be distinguished byTLC as well as by ³¹P-NMR: TLC (silica gel, EtOAc/n-heptane=1:2, +1%Et₃N): R_(f)(2 diastereomeres)=0.20; 0.27

331.3 mg (55.4% of the theoretical yield) of a white foaming residue isobtained. ³¹P-NMR (CD₃Cl) δ (ppm): 149.65, 148.59 MS (EI): 303 (DMT⁺),1003.1 (M+Na⁺)

EXAMPLE 3 Synthesis of 5-O-(4,4′-dimethoxytrityl)-(1,2)-dithian-4-ol

A solution of I₂ in DCM (9.4 mmol I₂ in 120 ml DCM) is added to asolution of 2 g (1.88 mmol)3-O-(4,4′-dimethoxytrityl)-1,4-bis-(4,4′-dimethoxitrityl)sulfanyl-butan-2-oland 5 ml pyridine in 100 ml DCM at room temperature. After 10 min ofstirring, 200 ml of a 0.5 N Na₂S₂O₃ solution is added. The layers areseparated, the organic phase extracted three times with H₂O and thecombined organic phases dried with Na₂SO₄. The solvent is evaporated andthe remaining foam purified by silica gel chromatography (eluent:gradient of 10-30% EtOAc in n-heptane in presence of 1% Et₃N). TLC(silica gel, EtOAc/n-heptane=1:2, +1% Et₃N): R_(f)(2diasteromeres)=0.31; 0.40.

710.7 mg (83.6% of the theoretical yield) of a yellowish oil isobtained. ¹³C-NMR (CD₃Cl) δ (ppm): 32.76 (C-6), 41.83 (C-3), 55.24(OCH₃), 67.21 (C-5), 72.32 (C-4), 87.25 (C_(q) DMT), 113.16, 126.64,127.89, 129.39, 130.61, 136.94, 145.14, 158.09 (C_(arom) DMT). ¹H-NMR(CD₃Cl) δ (ppm): 2.75 (m, 2H, CH₂-6), 3.03 (m, 2H, CH₂-3), 3.69 (m. 2H,H-4, H-5), 3.79 (s, 3H, OCH₃), 6.83-7.52 (m, 13H_(arom) DMT). MS(electrospray ionisation in MeOH): 303 (DMT⁺), 477 (M+Na⁺).

EXAMPLE 4 Synthesis of5-O-(4,4′-dimethoxytrityl)-(1,2)-dithian-4-O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite

A schematic overview of the synthesis is shown in FIG. 1.

In an argon atmosphere 500 mg (1.1 mol) of5-O-(4,4′-dimethoxytrityl)-(1,2)-dithian-4-ol is dissolved in 8 ml ofanhydrous DCM. The solution is cooled with ice and 753.2 μl (4.4 mmol)of N,N′-diisopropylethylamine is added dropwise while stirring. Using asyringe 295 μl (1.32 mmol) ofchlor-(2-cyanoethoxy)(diisopropylamino)-phosphine is added dropwise.After 1 hour of stirring at room temperature the reaction mixture isdiluted with 50 ml DCM and is extracted once with a 1 mol/l NaHCO₃solution and once with a saturated sodium chloride solution. The organicphase is dried with Na₂SO₄, filtered and concentrated. For purificationof the raw material silica gel chromatography is used (eluent: gradientfrom 5-15% EtOAc in n-heptane in presence of 1% Et₃N). Bothdiastereomeres can be distinguished by TLC as well as by ³¹P-NMR: TLC(silica gel, EtOAc/n-heptane=1:2, +1% Et₃N): R_(f)(2diastereomeres)=0.31; 0.40

459.6 mg (63.8% of the theoretical yield) of the desired product isobtained as a yellowish oil. ³¹P-NMR (CD₃Cl) δ (ppm): 148.33, 150.19. MS(EI): 303 (DMT⁺), 655 (M), 677 (M+Na⁺)

EXAMPLE 5 Synthesis of1,4-bis-(4,4′-dimethoxitrityl)sulfanyl-butan-2,3-diol

In an argon atmosphere 2.2 g (14.3 mmol) of 1,4-dithio-butan-2,3-diolare dissilved by stirring in 40 ml anhydrous pyridine in around-bottomed 250 ml flask. 9.93 g (29.3 mmol) of DMT-Cl(4,4′-Dimethoxytrityl Chloride) is added to the clear solution. After 2hours stirring at room temperature 2 ml MeOH is added and the mixture isstirred a further 5 min. The solvent is evaporated, the residue isdissolved in 50 ml DCM and is extracted once with a 1 mol/l NaHCO₃solution and once with a NaCl solution. The organic phase is dried withNa₂SO₄, filtered and concentrated. The remaining foam is purified bysilica gel chromatography (eluent: gradient of 10-30%ethylacetate/n-heptane in presence of 1% Et₃N). The product containingfractions are collected and the solvent is evaporated to dryness undervacuum. TLC (silica gel, EtOAc/n-heptane=1:2, +1% Et₃N): R_(f)=0.35.

8.66 g (79.8% of the theoretical yield) of a white, foaming residue isobtained. ¹³C-NMR (CD₃Cl) δ (ppm): 34.83 (C-1, C-4), 55.20 (OCH₃ DMT),65.94 (C-2, C-3), 71.70 (C_(q) DMT), 113.23, 126.61, 127.95, 129.40,130.62, 136.96, 145.14, 158.09 (C_(arom) DMT). ¹H-NMR (CD₃Cl) δ (ppm):2.01 (m, 2H, OH-2, OH-3), 2.35 (m, 4H, C-1, C-4), 3.05 (m, 2H, C-2,C-3), 3.73 (s, 12H, OCH₃), 6.78-7.45 (m, 26H_(arom) DMT).

EXAMPLE 6 Synthesis of3-O-(9-fluorenylmethoxycarbonyl)-1,4-bis-(4,4′-dimethoxitrityl)sulfanyl-butan-2-ol

In an argon atmosphere 407 mg (1.58 mmol) of9-Fluorenylmethylchloroformate is added to a solution of 1 g (1.32 mmol)of 1,4-bis-(4,4′-dimethoxitrityl)sulfanyl-butan-2,3-diol in 10 mlanhydrous pyridine. The reaction mixture is stirred overnight at roomtemperature. Then 500 μl MeOH is added and stirred for 10 min. Afterconcentration of the solvent the residue is dissolved in 30 ml DCM andextracted once with a NaCl solution. After the drying of the organicphase with Na₂SO₄ and evaporation of the solvent, the raw material ispurified by silica gel chromatography (eluent:ethylacetate/n-heptane=3:1). TLC (silica gel, EtOAc/n-heptane=1:2):R_(f)=0.25.

303 mg (23.4% of the theoretical yield) of a white foaming residue isobtained. ¹³C-NMR (CD₃Cl) δ (ppm): 31.68 (C-4), 35.06 (C-1), 55.20 (OCH₃DMT), 50.36 (CH-Fmoc), 65.31 and 65.45 (C-2 and C-3), 66.77 (CH₂-Fmoc),70.38 (2S-C-DMT), 113.23, 120.05, 124.70, 127.06, 127.6, 128.08, 129.22,136.24, 136.82, 141.51, 143.31, 144.35, 145.01, 154.36, 158.10, 158.3(C_(arom) DMT and Fmoc). ¹H-NMR (CD₃Cl) δ (ppm): 2.10-2.45 (m, 4H,CH₂-1, CH₂-4), 3.73 (d, 12H, OCH₃ DMT), 3.98-4.35 (m, 2H, H-3, H-2),6.78-7.82 (m, 34H, H_(arom) DMT and Fmoc). MS (electrospray ionisation):303.2 (DMT⁺), 1283.3 (M+Na⁺)

EXAMPLE 7 Synthesis of5-O-(9-fluorenylmethoxycarbonyl)-(1,2)-dithian-4-ol

A solution of I₂ in DCM (6.25 mmol I₂ in 60 ml DCM) is added to asolution of 1.23 g (1.25 mmol)3-O-(9-fluorenylmethyloxycarbonyl)-1,4-S,S′-bis-(4,4′-dimethoxitrity)-butan-2-olin 50 ml DCM at room temperature. After 10 min 100 ml of 0.5 N Na₂S₂O₃solution is added during rapid stirring. The layers are separated, theorganic phase is extracted three times with H₂O and the combined organicphases are dried with Na₂SO₄. The solvent is evaporated and theremaining foam is purified by silica gel chromatography (eluent:gradient of 10-30% EtOAc in n-heptane). TLC (silica gel,EtOAc/n-heptane=1:2): R_(f)=0.20.

212 mg (45.3% of the theoretical yield) of a yellow oil is obtained.¹³C-NMR (CD₃Cl) δ (ppm): 34.51 (C-6), 41.86 (C-3), 46.74 (CH-Fmoc),65.18 (CH₂-Fmoc), 70.11 (C-5), 72.21 (C-4), 120.06, 125.07, 127.59,127.98, 141.33, 143.18 (C_(arom) Fmoc) ¹H-NMR (CD₃Cl) δ (ppm): 2.98 (m,2H, CH₂-6), 3.09 (m, 2H, CH₂-3), 3.72 (m, 2H, H-4, H-5), 7.28-7.80 (m,8H_(arom) Fmoc).

EXAMPLE 8 Synthesis of3-O-(9-fluorenylmethoxycarbonyl)-1,4-bis-(4,4′-dimethoxitrityl)sulfanyl-butan-2-O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite

In an argon atmosphere 460 mg (0.47 mmol) of3-O-(9-fluorenylmethoxycarbonyl)-1,4-bis-(4,4′-dimethoxitrityl)sulfanyl-butan-2-olis dissolved in 5 ml of anhydrous DCM. 322 μl (1.88 mmol) ofN,N′-diisopropylethylamine is added to the ice cooled solution dropwisewhile stirring. 136.5 μl (0.61 mmol) ofchlor-(2-cyanoethoxy)(diisopropylamino)-phosphine is added dropwiseusing a syringe. After 1 hour of stirring at room temperature thereaction mixture is diluted with 50 ml DCM and is extracted once with a1 mol/l NaHCO₃ solution and once with a saturated sodium chloridesolution. The organic phase is dried with Na₂SO₄, filtered andconcentrated. For purification of the raw material silica gelchromatography is used (eluent: gradient from 5-20% EtOAc in n-heptanein presence of 1% Et₃N). Both diastereomeres can be distinguished by TLCas well as by ³¹P-NMR: TLC (silica gel, EtOAc/n-heptane=1:2, +1% Et₃N):R_(f)(2 diastereomeres)=0.26; 0.29

270 mg (48.7% of the theoretical yield) of a white foaming residue isobtained. ³¹P-NMR (CD₃Cl) δ (ppm): 149.35, 150.29.

EXAMPLE 9 Synthesis of5-O-(9-fluorenylmethoxycarbonyl-(1,2)-dithianyl-4-O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite

In an argon atmosphere 500 mg (1.33 mmol) of5-O-(9-fluorenylmethoxycarbonyl)-(1,2)-dithianyl-4-ol is dissolved in 5ml of anhydrous DCM. 914 μl (5.32 mmol) of N,N′-diisopropylethylamineare added dropwise to the ice cooled solution while stirring. 387 μl(1.73 mmol) of chlor-(2-cyanoethoxy)(diisopropylamino)-phosphine isadded dropwise using a syringe. After 1.5 hour of stirring at roomtemperature the reaction mixture is diluted with 50 ml DCM and isextracted once with a 1 mol/l NaHCO₃ solution and once with a saturatedsodium chloride solution. The organic phase is dried with Na₂SO₄,filtered and concentrated. For purification of the raw material silicagel chromatography is used (eluent: gradient from 5-20% EtOAc inn-heptane in presence of 1% Et₃N). Both diastereomeres can bedistinguished by TLC as well as by ³¹P-NMR: TLC (silica gel,EtOAc/n-heptane=1:2, +1% Et₃N): R_(f)(2 diastereomeres)=0.22; 0.3

295 mg (48.7% of the theoretical yield) of a white foaming residue isobtained. ³¹P-NMR (CD₃Cl) δ (ppm): 149.04, 150.32

EXAMPLE 10 Solid Phase Synthesis of Thiol Modified Oligonucleotides bythe Phosphoramidite Method Using the Compound as Described in Example 4

The synthesis of the oligodesoxyribonucleotides was carried out in a 1μmol synthesis scale using the solid phase phosphoramidite techniquewith an automated DNA/RNA synthesizer model 384 B (Applied Biosystems)on ®CPG (Controlled Pore Glass), on which the first nucleoside unit wasattached by the 3′ end. For this the synthesis instrument is for examplemounted with reaction columns which are filled with a carrier materialpreloaded with a nucleobase. In a first reaction step the 5′OHprotecting group (4,4′-dimethoxytrityl) is cleaved by treatment with asolution of 2% dichloracetic acid in dichlormethane. After washing thecolumn with acetonitril the coupling of the next building block whichcan be the modified amidite of example 4 is achieved at the free 5′—OHfunction by activation with tetrazole in acetonitril. For incorporationof the modified amidite respectively a 10 min double coupling step isused. The existing still trivalent P atom is transferred after renewedwashing into the natural pentavalent phosphate by oxidation with asolution of Iodine in THF/Lutidin/H₂O. The following capping step byacetic anhydride/1-methylimidazole blocks free 5′—OH groups byacetylation. Thus the building of failure sequences are suppressed.After washing the synthesis cycle starts over again with renewedcleavage of the 5′-O-dimethoxytrityl protecting group. In this way themodified oligonucleotide is built up. The last DMT group was notcleaved. After completed synthesis the oligonucleotide bound to thecarrier was set free by treatment with concentrated ammoniac solution inwater. The protecting groups at the heterocycles were removed in thesame solution within 16 h at 37° C. The samples were concentrated toapprox. 200 μl in vacuum and purified by HPLC.

EXAMPLE 11 Solid Phase Synthesis of Thiol Modified Oligonucleotides bythe Phosphoramidite Method Using the Compound as Described in Example 9

The oligomer synthesis takes place as described in example 10. For thedeprotection of the Fmoc group the oligonucleotide at the carrier istreated with a solution of 0.5 mol/l DBU in acetonitril (4×1 ml 0.5mol/l DBU/ACN in 2 min). The work up procedure also follows that inexample 10.

EXAMPLE 12 Solid Phase Synthesis of Thiol Modified Oligonucleotides bythe Phosphoramidite Method Using the Compound as Described in Example 2

The oxidation step is carried out using a 0.1 mol/l iodine solution withextended reaction times (1 min). The further synthesis cycle and thework up procedure of the oligonucleotides is as described in example 10.

EXAMPLE 13 HPLC Purificaton of Trityl Protected Oligonucleotides

In the first purification step the DMT protected oligomers are purifiedby HPLC with a RP-C18 silica gel column (eluent: 0.1 mol/ltriethylammoniumacetate buffer, acetonitril). The oligomers were treatedwith 100 μl of an 80% acetic acid solution and shaken for 20 min at roomtemperature. 100 μl H₂O und 60 μl 3 mol/l NaAc solution was added tothat solution. The oligonucleotides were treated with 1.5 ml EtOH andcompletely precipitated at −20° C. (20 min). After centrifugation anddecanting of the ethanol, the pellet is dried in vacuum. Thecharacterisation of the oligomers was effected using MALDI-TOF MS. Table1 shows the retention times of the synthesized oligonucleotides.

TABLE 1 retention oligomer (5′->3′-direction) times (min) (sequence)with DMT oligonucleotide 1: 5′-XAGG TGA CTG TGT TAT CCG CA-3′ 10.05oligonucleotide 2: 5′-XXAGG TGA CTG TGT TAT CCG CA-3′ 11.30oligonucleotide 3: 5′-XXXAGG TGA CTG TGT TAT CCG CA-3′ 11.72 where X iscompound 4 5′-XT10-3′ 21.12 5′-XXT10-3′ 21.52 where X is compound 2

EXAMPLE 14 Experiments for Immobilization of Oligonucleotides 1, 2 and 3According to Example 13

Preparation of Single-strand DNA Monolayer

a) Cleaning of Au Electrodes

To remove impurities on the gold surface the gold covered glas slideswere immersed in a mixture (3:1) of concentrated sulfuric acid andhydrogen peroxide solution (30%) for 30 seconds. The electrodes wererinsed thoroughly with deionized water and placed in pure ethanol for 15minutes.

b) Immobilization of Oligonucleotides 1, 2 and 3 on Gold Surfaces

Oligonucleotides were immobilized on gold surfaces overnight by selfassembly from 30 μM solutions in 500 mM potassium phosphate buffer (pH7.0). After adsorption the electrodes were rinsed thoroughly withpotassium phosphate buffer.

Surface Plasmon Resonance spectroscopy (SPR) was used to examine theeffectiveness of immobilization. SPR is highly sensitive to changes ofrefractive index at the metal interface that are a byproduct ofadsorption and desorption processes. The shifting ΔΘ of the resonanceangle is directly proportional to mass increase or decrease at thesurface. Experiments were carried out with a Biosuplar II (fromAnalytical μ-Systems, Regensburg, Germany).

FIG. 2 illustrates the SPR kinetics of the immobilization ofoligonucleotides oligo 1 (▪), oligo 2 (▴) and oligo 3 (●) onto a goldsurface (c=30 μM in 500 mM potassium phosphate buffer, pH 7). It can beseen from the plot that oligonucleotides 1, 2 and 3 show roughlyidentical kinetic behaviour for the immobilization process. The amountadsorbed on the surface is less than that of comparable oligonucleotideswith a single thiol anchor (□) (H₂N—C₆-TCG TCA CTG TCA GTG TCAGA-[C₃—S—S—C₃—OH] with C₃═(CH₂)₃ and C₆═(CH₂)₆), which reflects thehigher spacial requirement per DNA strand.

c) Coadsorption of Alkane Thiols

The oligonucleotide-modified gold surface was treated with short-chainalkane thiols in order to put the DNA in a more upright position and topassivate gaps on the surface. This coadsorption was carried out for 30minutes with a 1 mM solution of the corresponding thiol (propane thiolor 3-hydroxy propane thiol) in the above-mentioned potassium phosphatebuffer containing 1% ethanol followed by a thorough rinsing withpotassium phosphate buffer.

Stability Tests:

The stability of immobilization was checked by incubating the surfaceswith phosphate buffer (pH 9) and applying conditions for dehybridization(2 mol/l NaOH) under SPR control. FIG. 3 illustrates a stability testfor a monolayer of oligonucleotide 1 coadsorbed with propanethiol. Atthe point in time t=0 the oligonucleotide monolayer coadsorbed withpropanethiol is subjected to 500 mM phosphate buffer at pH 7. At t=1 hthe buffer was replaced with 500 mM phosphate buffer at pH 9. Theresonance angle is shifted as a result of the higher refractive index ofthis buffer. Subsequently at t=2 h, the buffer was changed back to theoriginal 500 mol/l phosphate buffer at pH 7 with the resonance anglealso returning to its initial value. The amount of substance on thesurface did not change during the treatment with buffer at pH 9. At t=5h the buffer was replaced by 2 mol/l NaOH and finally at t=5.5 h againchanged back to phosphate buffer, pH 7. Also when subjected to theseconditions, the monolayer remains stable without any loss of material.

Verification of Hybridizability

In order to check the hybridizability, the above oligonucleotidemonolayer was hybridized with a complementary strand double-labeled withferrocenyl acetic acid ([FcAc—Y]₂—C GGA TAA CAC AGT CAC CT; Y=AminoIntroducing Reagent with C3-spacer; Chemgene). The hybridization wasperformed by incubation of the monolayer with a 100 mmol/l sodiumsulfate solution containing 1 μmol/l of complementary strand heated to95° C., followed by cooling down over a period of at least 2 hours.

Square Wave and Cyclic voltammetric methods were employed forelectrochemical characterization. Both methods can be used for thedetection of surface-bound redox label (here ferrocenyl acetic acid).

With square wave voltammetry a linear voltage ramp is superimposed on asquare wave potential at a frequency f and an amplitude E^(˜)(in theexample f=10 Hz, E^(˜)=20 mV rms) and the current is detected at the endof every pulse. Through this the capacitive charging current isvirtually eliminated, resulting in a voltammetric peak. A relativecomparison is easily done, whereas absolute quantification isunproblematic.

With cyclic voltammetry a triangular voltage wave is driven and theresulting current is detected. Distinctive capactive charging currentscan complicate the quantification of faradayic currents. However, thenumber of transferred charges and thus the number of redox labels can bedetermined by the integration of peak areas.

Using the square wave voltammetry described above it was checked whetherthe redox label of the hybridized complementary strand could bedetected. The figures 4.1 and 4.2 illustrate the square wavevoltammogram (f=10 Hz, E^(˜)=20 mV rms) for the oligonucleotidemonolayer 1-3 coadsorbed with propanethiol (4.1) andhydroyx-propanethiol (4.2) and hybridized with the redox labeledcomplementary strand. All monolayers show a distinct peak at +0.23 V (vsAg/AgCl/3M KCl), which is caused by oxidation of the ferrocenyl aceticacid on the complementary strand and indicates a successfulhybridization of the monolayer. However the peak currents andconsequently the hybridization efficiency varies depending on thecoadsorption and oligonucleotide applied.

Cyclic voltammetry was used for quantification. By integration of thecyclic voltammograms (v=500 mV/s) in FIGS. 4.3 and 4.4 the number ofredox labels and thus the surface concentration Γ of the complementarystrand (roughness factor=2, number of labels per target=2) is determinedand plotted in FIG. 5. The surface concentration Γ is in directproportion to the hybrizability of the oligonucleotide monolayer.

Among the three oligonucleotides in example 13, oligonucleotide 2 showsthe best hybridizability. The hybridizability of the ssDNA monolayercoadsorbed with hydroxypropanethiol is thus significantly higher ascompared with the respective monolayer coadsorbed with propanethiol. Thesurface coverage is between 1·10⁻¹²−7·10¹² mol/cm² and is thus in arange that has already been determined by other groups (Herne T. M.,Tarlov M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920).

All the above electrochemical experiments were carried out in athree-electrode setup with the gold working electrode to be analyzed, aPt counter electrode and a reference electrode (Ag/AgCl/3M KCl) using anAutolab 12 potentiostat (Ecochemie, Netherlands).

EXAMPLE 15

2 mmol of p-tosyl chloride is added to a solution oftrans-1,2-dithiane-4,5-diol (2 mmol in anhydrous pyridine). Afterstirring for 2 hours at room temperature the solvent is evaporated. 0.5eq ethylene diamine in DMF is stirred in the presence of NaH for 10 minat room temperature. Compound 10 (see FIG. 6) is dissolved in DMF andadded to the ethylendiamin solution above. The reaction mixture isstirred under reflux for 6 hours. The desired product 11 (see FIG. 6) isisolated by silica gel chromatography and reacted with 1 eq DMT-Cl inpyridine obtaining product 12 (see FIG. 6). The DMT protected product ispurified by silica gel chromatography (eluent: ethylacetate/n-heptane inpresence of 1% Et₃N). 1 mmol of product 12 (see FIG. 6) is dissolved in10 ml of anhydrous DCM in an argon atmosphere. The solution is cooled inan ice bath and 4 eq of N,N′-diisopropylethylamine is added dropwisewhile stirring. Using a syringe, 1.2 eq ofchlor-(2-cyanoethoxy)(diisopropylamino)-phosphine is added dropwise.After 1.5 hours of stirring at room temperature the reaction mixture isdiluted with DCM and worked up with standard methods. For purificationof product 13 (see FIG. 6) silica gel chromatography is used (eluent:ethylacetate/n-heptane in presence of 1% Et₃N).

EXAMPLE 16

1 g (8 mmol) of 2,3-dimercapto-1-propanol is dissolved in ACN andoxidized by air (oxygen). The corresponding cyclic compound 14 (see FIG.7) is reacted with MsCl (Mesylchloride) in the presence of Et₃N. After 2hours stirring at room temperature the solvent is evaporated in a rotaryevaporator. A solution of 1 eq MMT-Ethylendiamine is treated with NaH inDMF. After 30 min stirring at room temperature 1 eq of compound 14 (seeFIG. 7) in DMF is added to the MMT-Ethylendiamine solution above. Thereaction mixture is stirred under reflux for 4 hours. The solvent isreduced with a rotary evaporator, the residue is worked up with standardmethods and purified with silica gel chromatography.

The MMT group of compound 16 (see FIG. 7) is cleaved with an acidicsolution (2% DCA in DMF). 1 eq of compound 17 (see FIG. 7) is dissolvedin DMF and reacted withN-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-L-Lysine(compound 18 (see FIG. 7)) in presence of HOBt and DCC. The reaction iscarried out overnight at room temperature. The solvent is evaporated andthe residue is worked up with standard methods and purified with silicagel chromatography using ethylacetate/n-heptane as eluent.

The FMOC group of compound 19 (see FIG. 7) is cleaved selectively with asolution of 500 mmol/l DBU in ACN. The free amino group of product 20(see FIG. 7) reacts with BrCH₂CH₂COOH to obtain product 21 (see FIG. 7)as the main product. The raw material is purified with silica gelchromatography using ethylacetate/n-heptane as eluent.

EXAMPLE 17

1 eq each of compund 22 (see FIG. 8), DCC and HOBt is added to asolution of 1 g (4.7 mmol) of 1,3,5-Benzene-tricarboxylic acid inanhydrous DMF. The reaction mixture is stirred overnight at roomtemperature. The solvent is concentrated and the desired product 23 (seeFIG. 8) is isolated with silica gel chromatography. Compound 23 (seeFIG. 8) is dissolved in DMF and reacts with Fmoc-ethylendiamine in thepresence of DCC and HOBt. After reducing the solvent in a vacuum, theraw product is purified with silica gel chromatography usingethylacetate/n-heptane as eluent.

EXAMPLE 18

1.2 eq of Fmoc-Cl ((9-Fluorenylmethyl)-chloroformate) are added to asolution of 2 g (13 mmol) of 3,5-Diamino benzoic acid in 40 ml anhydrouspyridine. After 2 hours stirring at room temperature the solvent isevaporated and the raw product is purified with silica gelchromatography using ethylacetate/n-heptane as eluent. Compound 25 (seeFIG. 9) is dissolved in a mixture of ACN and dioxane and reacted withlipoic acid N-Hydroxy-succinimide ester. The reaction mixture is stirredovernight at room temperature. The solvent is removed with a rotaryevaporator and the residue is dissloved in DCM and worked up withstandard methods. The raw product is purified with silica gelchromatography using ethylacetate/n-heptane as eluent to isolate thedesired product 27 (see FIG. 9).

1. 3,4-disubstituted-1,2-dithiocyclohexane compounds of the formula

wherein protecting-group-Y¹ is protecting-group-NH,protecting-group-NR⁴, protecting-group-O, CONH-protecting-group,protecting-group-OOC, protecting -group-S—S, —CH (protecting-group-O )₂,or protecting-group-S, wherein protecting-group is chosen from the groupconsisting of triphenylmethyl-, t-butoxycarbonyl-,benzyl-,2,4-dinitrophenyl-, 9-fluorenylmethoxycarbonyl-,allyloxycarbonyl-, benzyloxymethyl-, 4-azidobenzyloxycarbonyl-,acetamidomethyl-, 1-adamantyl-, 1-adamantyloxycarbonyl-, anisyl,benzamidomethyl-, biphenyldimethylsilyl-,2,4-dimethylthiophenoxycarbonyl-,1-methyl-1-(4-biphenyl)ethoxycarbonyl-, benzothiazole-2-sulfonyl-,t-butoxymethyl-, benzoyl-, benzyloxycarbonyl-, cyclohexan-1,2-diacetal-,cyclohexyl-, 2-(4,4-dimethyl -2,6-dioxocyclohexylidene)ethyl-,1-methyl-1-(3,5-dimethoxyphenyl )ethoxycarbonyl-,diethylisopropylsilyl-, 1,3-dithianyl-2-methyl-, 2,4-dimethoxybenzyl-,dithianylmethoxycarbonyl-, dimethoxytrityl-, p,p′-dinitrobenzhydryl-,2,4-dinitrophenyl-, 2,4-dimethylpent -3-yloxycarbonyl-,2-(diphenylphosphino)ethyl-, 9-fluorenylmethyl-, levulinoyl-,p-methoxybenzensulfonyl-, 2,6-dimethoxy-4-methoxybenzensulfonyl-,monomethoxytrityl-, methoxyphenylsulfonyl-, mesitylensulfonyl-,o-nitrobenzyl-, 2-[2-(benzyloxy)ethyl]benzoyl-,3-(3-pyridyl)allyloxycarbonyl-,2,2,5,7,8-pentamethylchroman-6-sulfonyl-, pivaloyloxymethyl-,t-butyldimethylsilyl-, t-butyldiphenylsilyl-,2,2,2-trichloro-1,1-dimethylethyl-, trifluoroacetyl-, triisobutylsilyl-,2,4,6-trimethylbenzyl-, trimethoxybenzyl-, p-toluensulfonyl- orbenzyloxycarbonyl-, Y² is —OH, —NH₂, —NHR³, —NR³R⁴, —COOH, —COCl,—COOCO—R⁶, —CONH₂, —CONHR³, —COOR³, —SO₃H, —SO₃Cl, —SH, —S—SR³, —CHO,—COR³, —C₂H₃O, halogen, —N₃, —NH—NH₂, —NCO, —NCS, wherein R³ is alkyl,heteroalkyl, aryl, cycloalkyl or a protecting group, wherein R³ in thegroups Y¹ and Y² may be equal or different, R⁴ is a protecting group,wherein R⁴ in the groups Y¹ and Y² may be equal or different, andwherein R⁴ and R³ may be equal or different, R⁶ is alkyl, heteroalkyl,aryl, or cycloalkyl, wherein R⁶ in the groups Y¹ and Y² may be equal ordifferent, wherein Y² may also be a group of the formula (II) or (III),

wherein X¹ is halogen or substituted amine, X² is alkyl, alkoxy, aryloxyor a cyano derivative of alkyl, alkoxy, aryloxy, X³ is halogen, aminogroup or oxygen and X⁴ is alkyl, alkoxy, aryloxy or X⁴ is H if X³ isoxygen.
 2. The compounds according to claim 1 whereinprotecting-group-Y¹ is protecting-group-OOC, protecting-group-O,protecting-group-S, protecting-group-NH or protecting-group-NR^(y) andY² is COOH, COOR^(x), OH, OR^(x), SH, SR^(x), NH₂, NHR^(x), NR^(x)R^(y),wherein R^(x) is a protecting group and R^(y) is alkyl having no morethan 15 C atoms, aryl having no more than 14 C atoms, cycloalkyl havingno more than 15 C atoms, heteroalkyl having no more than 15 C atoms, aprotecting group or a group of the formula (II) or (III).